Abstract A person, usually a child, dies of rabies every nine minutes globally. Current rabies virus (RABV) post- exposure prophylaxis (PEP) remains complicated and costly, requiring four to five doses of inactivated RABV- based vaccine plus rabies immune globulin (RIG). A single-dose vaccine that does not require expensive and often unavailable RIG would greatly increase the efficacy of RABV vaccination, reduce the cost associated with rabies prevention, and save lives. However, there is a gap in our understanding of how B cells are activated in response to RABV-based vaccination. For the last 35 years, B cells secreting IgG (but not IgM) were thought to be solely responsible for vaccine-induced protection against RABV infection via T cell-dependent responses in post-exposure settings. Furthermore, mechanisms by which B cells acquire RABV antigen were not previously known. Our laboratory has begun to unravel key attributes of rabies-specific B cell responses that contribute to the rapid induction of vaccine-induced virus neutralizing antibodies (VNAs). We showed that vaccine-induced T cell-independent (TI) and early extrafollicular T cell-dependent (TD) B cell responses, including neutralizing IgM, can limit dissemination of pathogenic RABV into the CNS, providing partial protection in mice. We also showed that free rabies particles migrate to subcapsular sinus macrophages in the draining lymph node, transferring RABV antigen directly to B cells. We aim to exploit these findings to develop a single-dose RABV vaccine regimen. Specifically, we hypothesize that a replication-deficient, matrix gene-deleted RABV-based vaccine (RABV-?M) with enhanced tropism to follicular B cells will result in antigen-specific B cells rapidly differentiating into IgM and IgG plasma cells, thereby increasing the kinetics, magnitude and quality of early B cell responses. In this proposal, our goals are to clone, recover and characterize RABV-?M-based vaccines with enhanced tropism to follicular B cells. Next, the new vaccines will be tested in well-described mouse models of rabies immunogenicity and post-exposure protection. Finally, we will confirm that the genetic modifications introduced into RABV-?M do not adversely affect safety and stability. In addition to expanding our knowledge of RABV immunity, key insights will be gained regarding how viral vaccines interact with and activate follicular B cells to induce rapid and potent immunity against viral infections. The major milestone at the completion of this project is the identification of a safe vaccination regimen that induces potent and rapid TI and extrafollicular TD B cell responses more effectively than the multi-dose human rabies vaccine, without the need for RIG. In summary, single-dose vaccine strategies capable of eliciting rapid and robust B cell responses will improve the efficacy of human rabies vaccines, reduce the cost associated with rabies prevention and save lives.